Project Summary/Abstract Every transplant organ experiences ischemia reperfusion injury (IRI). IRI evokes major intra-graft inflammatory immune responses, which augments graft immunogenicity, increases the rate of allograft rejection and markedly worsens solid organ transplantation outcomes. Reducing the deleterious effects of IRI- induced inflammatory responses has even greater clinical implications given the usage of marginal grafts as a common practice worldwide to address the shortage of organs. Nonetheless, the mechanisms by which IRI links the innate and adaptive immunity arms remain poorly explored. Our previous studies have shown that resident dendritic cells in the allografts (RDCA) play pivotal roles in linking innate and alloimmune responses. Data from our laboratory obtained using various transgenic models have led to the novel observation that increased production of IL-6 by RDCA exposed to oxidative stress leads to increased formation of CD4 alloreactive T cells in heart allografts and their respective draining lymph nodes (DLN). Intriguingly, the expression of peripheral node addressin (PNAd) molecules was markedly upregulated in the DLN of the respective ischemic organs. PNAd are adhesive molecules on the high endothelial venules of LN and play a central role in T cell homing to the LN. Use of systemic anti-IL6 therapy markedly reduced inflammatory responses in the ischemic allografts and prevented chronic rejection, which is the major obstacle to long-term acceptance of organ transplants. When we further examined the mechanism of RDCA induction by IRI, we found that IRI markedly increases autophagy in DC. Autophagy plays a key role in regulating the surface expression of MHC molecules on DC and release of inflammatory cytokines by DC. Finally, we have devised an innovative platform for the targeted delivery of immunoregulatory agents to organs and DLN to dampen the deleterious effects of IRI. With these data, the primary goal of this project is to investigate the mechanisms by which IRI enhances the immunogenicity of allografts and to identify novel protective strategies to minimize IRI, with the ultimate goal of promoting long-term allograft acceptance. In AIM 1, we will examine the mechanisms by which RDCA promotes IRI-induced alloimmunity. In AIM 2, we will examine the mechanism of suppression of IRI-induced alloimmunity by anti-IL-6 therapy. In AIM 3, we will develop DLN and heart allograft targeted delivery using nanocarriers of anti-IL6 to reduce IRI-induced alloimmunity without systemic immunosuppression.